Abstract
Protein ubiquitination is an important post-translational modification involved in several essential signalling pathways. It has different effects on the target protein substrate, i.e., it can trigger the degradation of the protein in the proteasome, change the interactions of the modified protein with its partners, or affect its localization and activity. In order to understand the molecular mechanisms underlying the consequences of protein ubiquitination, scientists have to face the challenging task of producing ubiquitinated proteins for structural characterization with X-ray crystallography and/or nuclear magnetic resonance (NMR) spectroscopy. These techniques require milligrams of homogeneous samples of high purity. The strategies proposed so far for the production of ubiquitinated proteins can be divided into two groups, i.e., chemical (or non-enzymatic) and enzymatic methodologies. In this review, we summarize the still very sparse examples available in the literature that describe successful production of ubiquitinated proteins amenable for biochemical and structural studies, and discuss advantages and disadvantages of the techniques proposed. We also give a perspective of the direction in which the field might evolve.
Highlights
Protein ubiquitination consists of the covalent attachment of the ε-amino group of a target protein lysine to the carboxylic group of the ubiquitin (Ub) C-terminal glycine via an isopeptide bond
Ub conjugation is performed by a cascade of events involving three classes of enzymes called Ub-activating enzymes (E1), Ub-conjugating enzymes (E2) and Ub ligases (E3), and is reversed by deubiquitinating enzymes (DUBs)
The authors managed to crystallize mono-ubiquitinated proliferating cell nuclear antigen (PCNA) and solve the X-ray structure of the covalent complex (Figure 5a). This result suggests that in vitro enzymatic ubiquitination is in principle a suitable approach to obtain a sample that is mono-ubiquitinated on a specific lysine residue
Summary
Protein ubiquitination consists of the covalent attachment of the ε-amino group of a target protein lysine to the carboxylic group of the ubiquitin (Ub) C-terminal glycine via an isopeptide bond. E2, E3 and DUBs, novel potential targets for anti-cancer drug discovery, are modified by Ub or Ub-like proteins in cells This strongly supports the importance of studying how ubiquitination modifies the structure and function of proteins and the role of poly-Ub chains of different linkages. Ub was attached enzymatically to the catalytic site of the E2 UbcH5 by mutating the catalytic C85 into a lysine [9] The purpose of these studies is not strictly directed towards the investigation of the effects of protein ubiquitination on a lysine residue, but to entrap enzymatic reaction intermediates, so we do not include such examples in this review. We apologize to colleagues whose contribution we have unintentionally omitted
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